ANP及其受体在大鼠耳蜗螺旋神经节的表达及分布特征
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摘要
耳蜗螺旋神经节（Spiral ganglion, SG）主要是由神经元和胶质细胞共同构成的外周感觉神经节组织，位于耳蜗蜗螺旋小管内，沿耳蜗轴螺旋式分布。螺旋神经元（Spiral ganglion neurons, SGNs）是位于螺旋神经节内的初级听觉传入神经元，将螺旋器感觉毛细胞感知的声信息向听觉中枢传递。根据形态学特征、周围神经支配形式和生理功能特点，可将SGNs分为两种类型：I型SGNs为胞体较大、有髓鞘的双极神经元，约占SGNs总数的90~95%，支配内毛细胞并对不同频率的声信号进行编码；II型SGNs为胞体较小、无髓鞘的假单极神经元，所占比例较少，支配外毛细胞和部分支持细胞，通过控制感觉上皮对声音的敏感性而反馈调节听觉感受。噪声、耳毒性药物、老龄化、基因突变等原发性因素，以及毛细胞死亡引发的继发性损伤都可造成SGNs变性、死亡，最终导致永久性听力损失（感音神经性聋）。探明在初级听觉神经元神经生理和突触传递过程中发挥重要调节作用的神经递质、调质以及神经活性物质的表达、功能、相互作用，及其在生理、病理条件下参与调节听觉信号转导的具体机制，有望为听力损伤的有效预防和治疗提供重要依据。
雪旺细胞（Schwann cells, SCs）和卫星细胞（Satellite glial cells, SGCs）是螺旋神经节内主要的胶质细胞。SCs与SGNs的神经纤维（神经突）紧密联系，并与I型SGNs的神经纤维形成髓鞘结构；SGCs紧密包绕SGNs胞体，共同构成一个功能性单元，并与I型SGNs胞体形成髓鞘结构。尽管对螺旋神经节内SGCs的具体功能仍然不清楚，但研究发现外周感觉神经节内的SGCs表达多种离子通道、神经递质受体以及神经营养因子来调节神经突触传递过程中的神经活性，并且形成一个与血脑屏障十分类似的选择性通透屏障来调节局部神经微环境和代谢，这些特点与中枢神经系统（CNS）的星形胶质细胞具有诸多相似之处。感觉神经节内神经元-SGCs间相互联系或相互作用（Neuron–SGCs communication），是调节神经突触传递、神经代谢平衡和神经保护作用的重要基础。因此，探寻调节螺旋神经节内SGN-SGC间相互联系的新靶点和新药物，有望为听力损伤的防治提供新的策略。
心钠素（Atrial natriuretic peptide,ANP）是由心房肌细胞合成、分泌的28个氨基酸多肽类激素，具有强大的排钠、利尿和血管舒张活性，是利钠肽家族中的一员，后者还包括B型利钠肽和C型利钠肽。ANP与两种定位于靶细胞膜上的高亲和功能受体相互作用，进而发挥维持血压、细胞外液体容积和心血管体液动态平衡等重要生物学效应。利钠肽A型受体（Natriuretic peptide receptor-A, NPR-A）具有鸟苷酸环化酶（guanylyl cyclase, GC）催化活性，可催化细胞内第二信使环磷酸鸟苷（cGMP）的形成，继而调节三种cGMP结合蛋白：①依赖于cGMP的蛋白激酶（PKG）、②依赖于cGMP的磷酸二酯酶（cGMP-regulated PDEs）、③环核苷酸门控离子通道（CNG），发挥多种生物学效应，包括调节血管紧张度、神经元兴奋性、跨上皮离子转运，以及视觉、嗅觉信号转导。利钠肽C型受体（Natriuretic peptide receptor-C, NPR-C）也称为清除受体，缺乏鸟苷酸环化酶催化结构域，通过受体介导的内化和降解作用调节局部利钠肽浓度。研究表明NPR-C可通过百日咳毒素敏感的抑制性鸟嘌呤核苷酸调节蛋白（Gi），或磷脂酶C途径，抑制环磷酸腺苷（cAMP）的合成，进而影响多种组织第二信使系统的信号转导，对cGMP的水平不造成影响。
除了心血管系统，ANP及其功能受体（NPR-A, NPR-C）在其他组织如肾脏、肾上腺、肺、脂肪组织、视网膜等也具有分布。此外，它们在CNS也具有表达，提示ANP可能作为一种重要的神经调质或神经肽参与神经和胶质功能的调节。关于ANP及其受体在鼠类内耳感觉以及分泌区域的分布也有报道，提示ANP可能作为一种局部激素参与内耳水电解质平衡的调节。既往研究证实ANP受体在豚鼠蜗轴组织以及大鼠螺旋神经节等耳蜗神经感觉区域具有表达，但ANP及其受体在螺旋神经节内的具体定位和功能仍然不清楚。基于以上思考，本研究中通过免疫组织化学（immunohistochemistry）、逆转录-聚合酶链式反应（RT-PCR）以及蛋白印迹（Westernblot）方法，检测ANP及其功能受体（NPR-A, NPR-C）在大鼠螺旋神经节内的细胞定位和在出生后不同发育时期大鼠螺旋神经节内的表达变化，并探讨ANP及其受体对于螺旋神经节功能可能具有的调节作用。本研究证实了ANP及其受体在出生后不同发育时期大鼠耳蜗螺旋神经节中具有表达，其主要定位于螺旋神经节的神经元和卫星细胞中；出生后各发育时期的大鼠耳蜗螺旋神经节具有合成、表达ANP及其受体的能力，ANP及其受体mRNA和蛋白表达量随着出生后发育发生变化。我们认为ANP可能作为内耳的一种重要的神经调质或神经肽，参与耳蜗螺旋神经节神经、胶质功能以及神经元-卫星细胞间相互联系的调节，进而影响内耳声信号转导和听觉系统发育。
第一部分：ANP及其受体在大鼠耳蜗螺旋神经节的表达
目的：明确ANP及其受体在大鼠耳蜗螺旋神经节的细胞定位、合成表达情况。
方法：取出生后（第7日，P7）大鼠耳蜗行冰冻切片和超薄切片，通过对耳蜗组织切片行免疫组织化学染色，激光共聚焦显微镜和透射电子显微镜检测ANP及其受体在螺旋神经节的细胞定位；取大鼠蜗轴组织，通过RT-PCR和Western blot方法，检测组织内ANP及其受体mRNA和蛋白的合成、表达情况。
结果：免疫组织化学方法（免疫荧光和免疫电镜）证实ANP及其受体在大鼠耳蜗螺旋神经节的两型SGNs以及SGCs具有共表达，其在SGNs的共表达具有不均一性（在部分SGNs的共表达相对较弱）。ANP主要定位在I、II型SGNs核周体胞质中，以及SGCs胞质中；NPR-A和NPR-C主要定位于I、II型SGNs以及SGCs质膜上和近质膜胞质中。以ANP、NPR-A和NPR-C的特异性上、下游引物，通过RT-PCR方法在大鼠蜗轴组织中扩增出其目的基因条带，证实大鼠蜗轴组织表达ANP及其受体的mRNA；以ANP、NPR-A和NPR-C的特异性抗体，通过Western blot方法在大鼠蜗轴组织中检测出其目的蛋白条带，证实大鼠蜗轴组织表达ANP及其受体的蛋白。
结论：ANP及其受体主要定位于大鼠耳蜗螺旋神经节的神经元和卫星细胞中，其mRNA和蛋白在蜗轴组织也具有表达。本研究证实了大鼠耳蜗螺旋神经节具有合成、表达ANP及其受体的能力，提示ANP可能通过与其受体作用，参与耳蜗螺旋神经节内神经与胶质功能，以及SGN-SGC间相互联系的调节。
第二部分：ANP及其受体在出生后不同发育时期大鼠耳蜗螺旋神经节的表达
目的：明确ANP及其受体在出生后不同发育时期大鼠耳蜗螺旋神经节的表达特征。
方法：取出生后不同发育时期（P0、P7、P14、P21和P28）大鼠耳蜗行冰冻切片，通过对耳蜗组织切片行免疫组织化学染色，激光共聚焦显微镜检测ANP及其受体在各发育时期大鼠螺旋神经节的表达情况；取各发育时期大鼠蜗轴组织，通过RT-PCR和Western blot方法检测组织内ANP及其受体mRNA和蛋白的合成、表达变化。
结果：免疫组织化学方法证实ANP及其受体在出生后各发育时期的大鼠螺旋神经节中均有表达；RT-PCR和Western blot方法在各发育时期大鼠蜗轴组织检测到ANP、NPR-A和NPR-C的mRNA和蛋白，证实其具有合成ANP及其受体的能力。ANP mRNA在大鼠蜗轴组织中的表达量于出生时（P0）为最低，随着发育迅速升高，在成年时期（P28）达最高水平。NPR-A mRNA在大鼠蜗轴组织中的表达量于出生时（P0）为最高，随着发育迅速降低，在成年时期（P28）达最低水平。NPR-C mRNA在大鼠蜗轴组织中的表达量于出生后（P0）为最高，随着发育逐渐降低且变化相对较小，在成年时期（P28）有轻微升高。ANP蛋白在大鼠蜗轴组织中的表达量于出生后（P0）开始升高，于出生后第7日（P7）为最高水平，随着发育迅速降低，在成年时期（P28）达最低水平。NPR-A蛋白在大鼠蜗轴组织中的表达量于出生时（P0）为最高，随着发育逐渐降低，在成年时期（P28）水平相对较低。NPR-C蛋白在大鼠蜗轴组织中的表达量于出生后（P0）为最高，随着发育逐渐降低，至出生后第14日（P14）听力形成时水平最低，随后在成年时期（P28）有轻微升高。
结论：ANP及其受体在出生后各发育时期大鼠耳蜗螺旋神经节中均具有表达，并且其mRNA和蛋白在大鼠蜗轴组织的表达量随出生后发育而发生改变。ANP及其受体蛋白在蜗轴组织中的表达量均随着出生后听觉发育成熟而相应下调，提示ANP可能通过与其受体作用，参与出生后螺旋神经节的神经和胶质发育，以及SGN-SGC间相互联系建立过程的调节。
The spiral ganglion (SG) is a peripheral cluster of both neurons and glial cellslocalized in Rosenthal’s canal, which coils around the cochlear modiolus. Spiral ganglionneurons (SGNs) are the primary afferent neurons in the spiral ganglion and play a criticalrole in hearing, transmitting primary acoustic information from the mechanosensory haircells in the organ of Corti to the higher auditory centers of the central nervous system(CNS). SGNs are divided into two subpopulations, type I and type II, according to theirdifferent morphology, synaptic connections and functions. The large, bipolar andmyelinated type I neurons innervate inner hair cells with their peripheral processes toprincipally encode the auditory signals and represent approximately90~95%of theafferent auditory neurons. The smaller, pseudomonopolar and non-myelinated type IIneurons innervate the outer hair cells and some of the supporting cells of the cochlea toprovide sensory feedback, controlling the sensitivity of the auditory epithelia to specific sound stimuli. Degeneration of SGNs, primarily resulting from noise trauma, ototoxicdrugs, infection, aging and genetic mutations, or secondarily occurring as a result of haircells loss, ultimately leads to permanent sensorineural hearing loss. Understanding theexpression, function and signaling interactions of the neurotransmitters, neuromodulatorsand other regulatory substances which affect neuronal physiology and neurotransmissionin primary auditory neurons, may offer insights into the mechanisms underlying normaland pathological states of hearing, and provide important clues for effective prophylacticand therapeutic treatment for hearing impairment.
The satellite glial cells (SGCs) and Schwann cells are the primary glial cells in thespiral ganglion. Schwann cells are found in close proximity to the neuronal processes oraxons where they from myelin sheaths around type I neuronal axons. Perineuronal SGCs,whose roles are still relatively poorly understood, envelop the somata of SGNs, andtogether they constitute functional units. Additionally, SGCs form loose myelin around thesomatic membrane of type I neurons. SGCs in the peripheral sensory ganglion share manyproperties with astrocytes in the CNS, as they not only express various ion channels,neurotransmitter receptors and trophic factors which modulate neuronal activity insynaptic transmission, but also form a partial diffusion barrier which regulates theneuronal environment, resembling the blood-brain barrier in the CNS. Consequently, theinteraction between neurons and SGCs (Neuron–SGC communication) can modulateneurotransmission, neuroprotection and neuronal homeostasis. Therefore, new strategiesaimed at manipulating communication between SGNs and SGCs within the spiralganglion have the potential to become promising pharmacological targets that facilitatenew and effective therapies for hearing impairment.
Atrial natriuretic peptide (ANP) is a28amino acid peptide with potent natriuretic,diuretic, and vasorelaxant activity which is predominantly synthesized and secreted by thecardiac atria, and is the first member of the natriuretic peptide family, which also includesbrain natriuretic peptide and C-type natriuretic peptide. ANP interacts with two specific,high affinity receptors, NPR-A and NPR-C, on the plasma membrane of target cells tomediate its physiological effects on the regulation of cardiovascular homeostasis,maintaining blood pressure and extracellular fluid volume. The natriuretic peptidereceptor-A (NPR-A) is coupled to the particulate guanylyl cyclase (GC) which catalyzesthe synthesis of the second messenger cyclic guanosine monophosphate (cGMP). cGMPmodulates the activity of specific effector molecules including cGMP-regulated isoforms of phosphodiesterases, cyclic-nucleotide-gated ion channels, and cGMP-dependent proteinkinases G, which in turn regulate diverse biological responses associated with blood vesseltone, neuronal excitability, transepithelial ion transportation, and the sensory transductionpathways underlying olfaction and vision. The natriuretic peptide receptor-C (NPR-C),which lacks the GC domain, contributes to the clearance of ANP and other natriureticpeptides from the circulation through receptor-mediated internalization and degradation. Anumber of studies have shown that NPR-C can also affect second messenger systems in avariety of tissues by inhibiting cyclic AMP (cAMP) production via a pertussistoxin-sensitive inhibitory guanine nucleotide regulatory protein (Gi), and by activatingphospholipase-C without affecting cGMP levels.
In addition to the cardiovascular system, the tissue-specific distribution and functionof ANP, NPR-A and NPR-C has been established in several tissues including kidney,adrenal, lung, adipose tissue and retina. Furthermore, ANP and its receptors have beenfound in the CNS, leading us to speculate that ANP may function as a neuromodulator orneuropeptide involved in neuronal and glial functions. Importantly, their presence in thesecretory and sensory compartments of the rodent inner ear is well-documented,suggesting ANP may act as a local hormone regulating the fluid and electrolyte balance inthe inner ear. ANP receptors have been localized to the cochlear modiolus of the guineapig and rat spiral ganglion, the neuronal region of the cochlea. However, little is knownregarding the localization and function of ANP and its receptors within the spiral ganglion.In our current study, we investigated the expression and localization of ANP and itsreceptors in the cochlear spiral ganglion of the postnatal rat by immunohistochemistry,reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis, withthe aim of identifying their cellular localizations, expression levels and potential functions.We showed here that ANP and its receptors were colocalized in both subtypes of SGNsand perineuronal SGCs within the rat spiral ganglion. Additionally, we analyzed thedifferential expression levels of both mRNA and protein of ANP and its receptors withinthe rat spiral ganglion during postnatal development. Collectively, our data provide directevidence for the presence and synthesis of ANP as well as its receptors in both neuronaland nonneuronal cells within the cochlear spiral ganglion, suggesting possible roles forANP in modulating neuronal and glial functions as well as neuron–SGC communicationwithin the spiral ganglion during auditory neurotransmission and development.
Part one: Expression and localization of atrialnatriuretic peptide and its receptors in the spiralganglion of the rat cochlea
[Objectives] To investigate the expression and cellular localization of ANP and itsreceptors in the spiral ganglion of the rat cochlea.
[Methods] All cochleae used in this investigation were obtained from postnatalSprague Dawley rats (postnatal day7, P7). To reveal the cellular localization of ANP andits receptors in the rat spiral ganglion, both the cochlear cryosections and ultrathin sectionswere subjected to immunohistochemistry, and then examined under a scanning laserconfocal microscope or a transmission electron microscope, respectively. To investigatethe expression of ANP and its receptors in the rat spiral ganglion, both mRNA and proteinproducts of ANP and its receptors were detected in the cochlear modiolus tissues byRT-PCR and Western blot analysis.
[Results] The co-localization of ANP and its receptors were found in both subtypes ofSGNs and in perineuronal SGCs by immunohistochemistry, while the heterogeneity ofimmunoreactivity of ANP and its receptors were less co-localizated in a sub-population ofSGNs. Specifically, ANP was predominantly immunoreactive in the perikaryal cytoplasm ofboth type I and type II SGNs and in the perinuclear cytoplasm of SGCs. The distribution ofNPR-A and NPR-C was rather similar, and they were predominantly immunoreactive in theplasma membrane and adjacent cytoplasm of both type I and type II SGNs as well assurrounding SGCs. Subsequently, the transcripts of ANP and its receptor were detected inthe cochlear modiolus tissues by RT-PCR using specific primer pairs for ANP, NPR-A andNPR-C. The protein products were also present in the modiolus tissues by Western blottingusing specific antibodies against ANP, NPR-A and NPR-C.
[Conclusions] We have demonstrated that ANP and its receptors colocalize in bothsubtypes of SGNs and perineuronal SGCs of the rat spiral ganglion. Furthermore, we haveconfirmed the expressions of ANP and its receptors in the rat spiral ganglion at both theprotein and mRNA level. Collectively, our research provides a direct evidence for thepresence and synthesis of ANP and its receptors in both neuronal and non-neuronal cells within the spiral ganglion of the rat cochlea, suggesting possible roles for ANP inmodulating neuronal and glial functions, as well as neuron–SGC communication withinthe spiral ganglion during auditory neurotransmission via its receptors.
Part two: Expression patterns of atrial natriureticpeptide and its receptors within the spiral ganglion ofthe rat cochlea during postnatal development
[Objectives] To investigate the expression profiles of ANP and its receptors withinthe spiral ganglion of the rat cochlea during postnatal development.
[Methods] All cochleae used in this investigation were obtained from postnatalSprague Dawley rats at five different ages (from postnatal day0to day28, P0-P28). Toreveal the cellular localization of ANP and its receptors in the rat spiral ganglion duringpostnatal development, the cochlear cryosections were subjected to immunohistochemistryand examined under a scanning laser confocal microscope. To investigate the expressionlevels of ANP and its receptors in the rat spiral ganglion, both mRNA and proteinproducts of ANP and its receptors were detected and analyzed in the cochlear modiolustissues by RT-PCR and Western blot analysis.
[Results] The co-localization of ANP and its receptors were confirmed within thespiral ganglion of the rat cochlea at various stages of postnatal development byimmunohistochemistry. Subsequently, the differential expressions of ANP and itsreceptors at both the protein and mRNA level were detected in the cochlear modiolustissues by RT-PCR and Western blotting. Specifically, ANP mRNA was expressed at thelowest level after birth but strongly increased to a maximum level at P28. NPR-A mRNAwas expressed at a maximum level after birth but gradually decreased to a minimum levelat P28. The expression of NPR-C mRNA was decreased at early postnatal developmentalstages but slightly increased in adulthood. In contrast to the qRT-PCR data, ANP proteinwas expressed at a maximum level at P7but sharply decreased to the lowest level at P28.NPR-A and NPR-C proteins were expressed at a maximum level after birth but graduallydeclined during postnatal development.
[Conclusions] We have demonstrated that ANP and its receptors colocalize in the spiral ganglion of the rat cochlea during postnatal development. Furthermore, we haveanalyzed the differential expression levels of both mRNA and protein of ANP and itsreceptors within the rat spiral ganglion during postnatal development. Collectively, ourresearch provides a direct evidence for the presence and synthesis of ANP and itsreceptors in the spiral ganglion of the rat cochlea during postnatal development. Theprotein expression levels of ANP and its receptors decreased with auditory development,suggesting possible roles for ANP in modulating neuronal and glial development, as wellas establishment of the neuron–SGC communication via its receptors.
雪旺细胞（Schwann cells, SCs）和卫星细胞（Satellite glial cells, SGCs）是螺旋神经节内主要的胶质细胞。SCs与SGNs的神经纤维（神经突）紧密联系，并与I型SGNs的神经纤维形成髓鞘结构；SGCs紧密包绕SGNs胞体，共同构成一个功能性单元，并与I型SGNs胞体形成髓鞘结构。尽管对螺旋神经节内SGCs的具体功能仍然不清楚，但研究发现外周感觉神经节内的SGCs表达多种离子通道、神经递质受体以及神经营养因子来调节神经突触传递过程中的神经活性，并且形成一个与血脑屏障十分类似的选择性通透屏障来调节局部神经微环境和代谢，这些特点与中枢神经系统（CNS）的星形胶质细胞具有诸多相似之处。感觉神经节内神经元-SGCs间相互联系或相互作用（Neuron–SGCs communication），是调节神经突触传递、神经代谢平衡和神经保护作用的重要基础。因此，探寻调节螺旋神经节内SGN-SGC间相互联系的新靶点和新药物，有望为听力损伤的防治提供新的策略。
心钠素（Atrial natriuretic peptide,ANP）是由心房肌细胞合成、分泌的28个氨基酸多肽类激素，具有强大的排钠、利尿和血管舒张活性，是利钠肽家族中的一员，后者还包括B型利钠肽和C型利钠肽。ANP与两种定位于靶细胞膜上的高亲和功能受体相互作用，进而发挥维持血压、细胞外液体容积和心血管体液动态平衡等重要生物学效应。利钠肽A型受体（Natriuretic peptide receptor-A, NPR-A）具有鸟苷酸环化酶（guanylyl cyclase, GC）催化活性，可催化细胞内第二信使环磷酸鸟苷（cGMP）的形成，继而调节三种cGMP结合蛋白：①依赖于cGMP的蛋白激酶（PKG）、②依赖于cGMP的磷酸二酯酶（cGMP-regulated PDEs）、③环核苷酸门控离子通道（CNG），发挥多种生物学效应，包括调节血管紧张度、神经元兴奋性、跨上皮离子转运，以及视觉、嗅觉信号转导。利钠肽C型受体（Natriuretic peptide receptor-C, NPR-C）也称为清除受体，缺乏鸟苷酸环化酶催化结构域，通过受体介导的内化和降解作用调节局部利钠肽浓度。研究表明NPR-C可通过百日咳毒素敏感的抑制性鸟嘌呤核苷酸调节蛋白（Gi），或磷脂酶C途径，抑制环磷酸腺苷（cAMP）的合成，进而影响多种组织第二信使系统的信号转导，对cGMP的水平不造成影响。
除了心血管系统，ANP及其功能受体（NPR-A, NPR-C）在其他组织如肾脏、肾上腺、肺、脂肪组织、视网膜等也具有分布。此外，它们在CNS也具有表达，提示ANP可能作为一种重要的神经调质或神经肽参与神经和胶质功能的调节。关于ANP及其受体在鼠类内耳感觉以及分泌区域的分布也有报道，提示ANP可能作为一种局部激素参与内耳水电解质平衡的调节。既往研究证实ANP受体在豚鼠蜗轴组织以及大鼠螺旋神经节等耳蜗神经感觉区域具有表达，但ANP及其受体在螺旋神经节内的具体定位和功能仍然不清楚。基于以上思考，本研究中通过免疫组织化学（immunohistochemistry）、逆转录-聚合酶链式反应（RT-PCR）以及蛋白印迹（Westernblot）方法，检测ANP及其功能受体（NPR-A, NPR-C）在大鼠螺旋神经节内的细胞定位和在出生后不同发育时期大鼠螺旋神经节内的表达变化，并探讨ANP及其受体对于螺旋神经节功能可能具有的调节作用。本研究证实了ANP及其受体在出生后不同发育时期大鼠耳蜗螺旋神经节中具有表达，其主要定位于螺旋神经节的神经元和卫星细胞中；出生后各发育时期的大鼠耳蜗螺旋神经节具有合成、表达ANP及其受体的能力，ANP及其受体mRNA和蛋白表达量随着出生后发育发生变化。我们认为ANP可能作为内耳的一种重要的神经调质或神经肽，参与耳蜗螺旋神经节神经、胶质功能以及神经元-卫星细胞间相互联系的调节，进而影响内耳声信号转导和听觉系统发育。
第一部分：ANP及其受体在大鼠耳蜗螺旋神经节的表达
目的：明确ANP及其受体在大鼠耳蜗螺旋神经节的细胞定位、合成表达情况。
方法：取出生后（第7日，P7）大鼠耳蜗行冰冻切片和超薄切片，通过对耳蜗组织切片行免疫组织化学染色，激光共聚焦显微镜和透射电子显微镜检测ANP及其受体在螺旋神经节的细胞定位；取大鼠蜗轴组织，通过RT-PCR和Western blot方法，检测组织内ANP及其受体mRNA和蛋白的合成、表达情况。
结果：免疫组织化学方法（免疫荧光和免疫电镜）证实ANP及其受体在大鼠耳蜗螺旋神经节的两型SGNs以及SGCs具有共表达，其在SGNs的共表达具有不均一性（在部分SGNs的共表达相对较弱）。ANP主要定位在I、II型SGNs核周体胞质中，以及SGCs胞质中；NPR-A和NPR-C主要定位于I、II型SGNs以及SGCs质膜上和近质膜胞质中。以ANP、NPR-A和NPR-C的特异性上、下游引物，通过RT-PCR方法在大鼠蜗轴组织中扩增出其目的基因条带，证实大鼠蜗轴组织表达ANP及其受体的mRNA；以ANP、NPR-A和NPR-C的特异性抗体，通过Western blot方法在大鼠蜗轴组织中检测出其目的蛋白条带，证实大鼠蜗轴组织表达ANP及其受体的蛋白。
结论：ANP及其受体主要定位于大鼠耳蜗螺旋神经节的神经元和卫星细胞中，其mRNA和蛋白在蜗轴组织也具有表达。本研究证实了大鼠耳蜗螺旋神经节具有合成、表达ANP及其受体的能力，提示ANP可能通过与其受体作用，参与耳蜗螺旋神经节内神经与胶质功能，以及SGN-SGC间相互联系的调节。
第二部分：ANP及其受体在出生后不同发育时期大鼠耳蜗螺旋神经节的表达
目的：明确ANP及其受体在出生后不同发育时期大鼠耳蜗螺旋神经节的表达特征。
方法：取出生后不同发育时期（P0、P7、P14、P21和P28）大鼠耳蜗行冰冻切片，通过对耳蜗组织切片行免疫组织化学染色，激光共聚焦显微镜检测ANP及其受体在各发育时期大鼠螺旋神经节的表达情况；取各发育时期大鼠蜗轴组织，通过RT-PCR和Western blot方法检测组织内ANP及其受体mRNA和蛋白的合成、表达变化。
结果：免疫组织化学方法证实ANP及其受体在出生后各发育时期的大鼠螺旋神经节中均有表达；RT-PCR和Western blot方法在各发育时期大鼠蜗轴组织检测到ANP、NPR-A和NPR-C的mRNA和蛋白，证实其具有合成ANP及其受体的能力。ANP mRNA在大鼠蜗轴组织中的表达量于出生时（P0）为最低，随着发育迅速升高，在成年时期（P28）达最高水平。NPR-A mRNA在大鼠蜗轴组织中的表达量于出生时（P0）为最高，随着发育迅速降低，在成年时期（P28）达最低水平。NPR-C mRNA在大鼠蜗轴组织中的表达量于出生后（P0）为最高，随着发育逐渐降低且变化相对较小，在成年时期（P28）有轻微升高。ANP蛋白在大鼠蜗轴组织中的表达量于出生后（P0）开始升高，于出生后第7日（P7）为最高水平，随着发育迅速降低，在成年时期（P28）达最低水平。NPR-A蛋白在大鼠蜗轴组织中的表达量于出生时（P0）为最高，随着发育逐渐降低，在成年时期（P28）水平相对较低。NPR-C蛋白在大鼠蜗轴组织中的表达量于出生后（P0）为最高，随着发育逐渐降低，至出生后第14日（P14）听力形成时水平最低，随后在成年时期（P28）有轻微升高。
结论：ANP及其受体在出生后各发育时期大鼠耳蜗螺旋神经节中均具有表达，并且其mRNA和蛋白在大鼠蜗轴组织的表达量随出生后发育而发生改变。ANP及其受体蛋白在蜗轴组织中的表达量均随着出生后听觉发育成熟而相应下调，提示ANP可能通过与其受体作用，参与出生后螺旋神经节的神经和胶质发育，以及SGN-SGC间相互联系建立过程的调节。
The spiral ganglion (SG) is a peripheral cluster of both neurons and glial cellslocalized in Rosenthal’s canal, which coils around the cochlear modiolus. Spiral ganglionneurons (SGNs) are the primary afferent neurons in the spiral ganglion and play a criticalrole in hearing, transmitting primary acoustic information from the mechanosensory haircells in the organ of Corti to the higher auditory centers of the central nervous system(CNS). SGNs are divided into two subpopulations, type I and type II, according to theirdifferent morphology, synaptic connections and functions. The large, bipolar andmyelinated type I neurons innervate inner hair cells with their peripheral processes toprincipally encode the auditory signals and represent approximately90~95%of theafferent auditory neurons. The smaller, pseudomonopolar and non-myelinated type IIneurons innervate the outer hair cells and some of the supporting cells of the cochlea toprovide sensory feedback, controlling the sensitivity of the auditory epithelia to specific sound stimuli. Degeneration of SGNs, primarily resulting from noise trauma, ototoxicdrugs, infection, aging and genetic mutations, or secondarily occurring as a result of haircells loss, ultimately leads to permanent sensorineural hearing loss. Understanding theexpression, function and signaling interactions of the neurotransmitters, neuromodulatorsand other regulatory substances which affect neuronal physiology and neurotransmissionin primary auditory neurons, may offer insights into the mechanisms underlying normaland pathological states of hearing, and provide important clues for effective prophylacticand therapeutic treatment for hearing impairment.
The satellite glial cells (SGCs) and Schwann cells are the primary glial cells in thespiral ganglion. Schwann cells are found in close proximity to the neuronal processes oraxons where they from myelin sheaths around type I neuronal axons. Perineuronal SGCs,whose roles are still relatively poorly understood, envelop the somata of SGNs, andtogether they constitute functional units. Additionally, SGCs form loose myelin around thesomatic membrane of type I neurons. SGCs in the peripheral sensory ganglion share manyproperties with astrocytes in the CNS, as they not only express various ion channels,neurotransmitter receptors and trophic factors which modulate neuronal activity insynaptic transmission, but also form a partial diffusion barrier which regulates theneuronal environment, resembling the blood-brain barrier in the CNS. Consequently, theinteraction between neurons and SGCs (Neuron–SGC communication) can modulateneurotransmission, neuroprotection and neuronal homeostasis. Therefore, new strategiesaimed at manipulating communication between SGNs and SGCs within the spiralganglion have the potential to become promising pharmacological targets that facilitatenew and effective therapies for hearing impairment.
Atrial natriuretic peptide (ANP) is a28amino acid peptide with potent natriuretic,diuretic, and vasorelaxant activity which is predominantly synthesized and secreted by thecardiac atria, and is the first member of the natriuretic peptide family, which also includesbrain natriuretic peptide and C-type natriuretic peptide. ANP interacts with two specific,high affinity receptors, NPR-A and NPR-C, on the plasma membrane of target cells tomediate its physiological effects on the regulation of cardiovascular homeostasis,maintaining blood pressure and extracellular fluid volume. The natriuretic peptidereceptor-A (NPR-A) is coupled to the particulate guanylyl cyclase (GC) which catalyzesthe synthesis of the second messenger cyclic guanosine monophosphate (cGMP). cGMPmodulates the activity of specific effector molecules including cGMP-regulated isoforms of phosphodiesterases, cyclic-nucleotide-gated ion channels, and cGMP-dependent proteinkinases G, which in turn regulate diverse biological responses associated with blood vesseltone, neuronal excitability, transepithelial ion transportation, and the sensory transductionpathways underlying olfaction and vision. The natriuretic peptide receptor-C (NPR-C),which lacks the GC domain, contributes to the clearance of ANP and other natriureticpeptides from the circulation through receptor-mediated internalization and degradation. Anumber of studies have shown that NPR-C can also affect second messenger systems in avariety of tissues by inhibiting cyclic AMP (cAMP) production via a pertussistoxin-sensitive inhibitory guanine nucleotide regulatory protein (Gi), and by activatingphospholipase-C without affecting cGMP levels.
In addition to the cardiovascular system, the tissue-specific distribution and functionof ANP, NPR-A and NPR-C has been established in several tissues including kidney,adrenal, lung, adipose tissue and retina. Furthermore, ANP and its receptors have beenfound in the CNS, leading us to speculate that ANP may function as a neuromodulator orneuropeptide involved in neuronal and glial functions. Importantly, their presence in thesecretory and sensory compartments of the rodent inner ear is well-documented,suggesting ANP may act as a local hormone regulating the fluid and electrolyte balance inthe inner ear. ANP receptors have been localized to the cochlear modiolus of the guineapig and rat spiral ganglion, the neuronal region of the cochlea. However, little is knownregarding the localization and function of ANP and its receptors within the spiral ganglion.In our current study, we investigated the expression and localization of ANP and itsreceptors in the cochlear spiral ganglion of the postnatal rat by immunohistochemistry,reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis, withthe aim of identifying their cellular localizations, expression levels and potential functions.We showed here that ANP and its receptors were colocalized in both subtypes of SGNsand perineuronal SGCs within the rat spiral ganglion. Additionally, we analyzed thedifferential expression levels of both mRNA and protein of ANP and its receptors withinthe rat spiral ganglion during postnatal development. Collectively, our data provide directevidence for the presence and synthesis of ANP as well as its receptors in both neuronaland nonneuronal cells within the cochlear spiral ganglion, suggesting possible roles forANP in modulating neuronal and glial functions as well as neuron–SGC communicationwithin the spiral ganglion during auditory neurotransmission and development.
Part one: Expression and localization of atrialnatriuretic peptide and its receptors in the spiralganglion of the rat cochlea
[Objectives] To investigate the expression and cellular localization of ANP and itsreceptors in the spiral ganglion of the rat cochlea.
[Methods] All cochleae used in this investigation were obtained from postnatalSprague Dawley rats (postnatal day7, P7). To reveal the cellular localization of ANP andits receptors in the rat spiral ganglion, both the cochlear cryosections and ultrathin sectionswere subjected to immunohistochemistry, and then examined under a scanning laserconfocal microscope or a transmission electron microscope, respectively. To investigatethe expression of ANP and its receptors in the rat spiral ganglion, both mRNA and proteinproducts of ANP and its receptors were detected in the cochlear modiolus tissues byRT-PCR and Western blot analysis.
[Results] The co-localization of ANP and its receptors were found in both subtypes ofSGNs and in perineuronal SGCs by immunohistochemistry, while the heterogeneity ofimmunoreactivity of ANP and its receptors were less co-localizated in a sub-population ofSGNs. Specifically, ANP was predominantly immunoreactive in the perikaryal cytoplasm ofboth type I and type II SGNs and in the perinuclear cytoplasm of SGCs. The distribution ofNPR-A and NPR-C was rather similar, and they were predominantly immunoreactive in theplasma membrane and adjacent cytoplasm of both type I and type II SGNs as well assurrounding SGCs. Subsequently, the transcripts of ANP and its receptor were detected inthe cochlear modiolus tissues by RT-PCR using specific primer pairs for ANP, NPR-A andNPR-C. The protein products were also present in the modiolus tissues by Western blottingusing specific antibodies against ANP, NPR-A and NPR-C.
[Conclusions] We have demonstrated that ANP and its receptors colocalize in bothsubtypes of SGNs and perineuronal SGCs of the rat spiral ganglion. Furthermore, we haveconfirmed the expressions of ANP and its receptors in the rat spiral ganglion at both theprotein and mRNA level. Collectively, our research provides a direct evidence for thepresence and synthesis of ANP and its receptors in both neuronal and non-neuronal cells within the spiral ganglion of the rat cochlea, suggesting possible roles for ANP inmodulating neuronal and glial functions, as well as neuron–SGC communication withinthe spiral ganglion during auditory neurotransmission via its receptors.
Part two: Expression patterns of atrial natriureticpeptide and its receptors within the spiral ganglion ofthe rat cochlea during postnatal development
[Objectives] To investigate the expression profiles of ANP and its receptors withinthe spiral ganglion of the rat cochlea during postnatal development.
[Methods] All cochleae used in this investigation were obtained from postnatalSprague Dawley rats at five different ages (from postnatal day0to day28, P0-P28). Toreveal the cellular localization of ANP and its receptors in the rat spiral ganglion duringpostnatal development, the cochlear cryosections were subjected to immunohistochemistryand examined under a scanning laser confocal microscope. To investigate the expressionlevels of ANP and its receptors in the rat spiral ganglion, both mRNA and proteinproducts of ANP and its receptors were detected and analyzed in the cochlear modiolustissues by RT-PCR and Western blot analysis.
[Results] The co-localization of ANP and its receptors were confirmed within thespiral ganglion of the rat cochlea at various stages of postnatal development byimmunohistochemistry. Subsequently, the differential expressions of ANP and itsreceptors at both the protein and mRNA level were detected in the cochlear modiolustissues by RT-PCR and Western blotting. Specifically, ANP mRNA was expressed at thelowest level after birth but strongly increased to a maximum level at P28. NPR-A mRNAwas expressed at a maximum level after birth but gradually decreased to a minimum levelat P28. The expression of NPR-C mRNA was decreased at early postnatal developmentalstages but slightly increased in adulthood. In contrast to the qRT-PCR data, ANP proteinwas expressed at a maximum level at P7but sharply decreased to the lowest level at P28.NPR-A and NPR-C proteins were expressed at a maximum level after birth but graduallydeclined during postnatal development.
[Conclusions] We have demonstrated that ANP and its receptors colocalize in the spiral ganglion of the rat cochlea during postnatal development. Furthermore, we haveanalyzed the differential expression levels of both mRNA and protein of ANP and itsreceptors within the rat spiral ganglion during postnatal development. Collectively, ourresearch provides a direct evidence for the presence and synthesis of ANP and itsreceptors in the spiral ganglion of the rat cochlea during postnatal development. Theprotein expression levels of ANP and its receptors decreased with auditory development,suggesting possible roles for ANP in modulating neuronal and glial development, as wellas establishment of the neuron–SGC communication via its receptors.
引文
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